Culture conditions have been devised in which fusion-blocked myoblasts, synchronized in M complete one round of synchronous division, blocking in the second G1 period. In this second, protracted G1, virtually the entire population (90%) initiates myosin synthesis. If, however, these cells, in their "terminal G1" are stimulated by a step-up to high growth medium, the population re-enters S. By double-labeling techniques (immunofluorescence and 3H-TdR incorporation) myosin-positive cells re-enter S with essentially the same frenquency as undifferentiated myoblasts. These studies will be extended to establish the fraction of cells which re-enter S when stimulated at different times during the terminal G1, and to determine wehther myosin synthesis continues in stimulated myocytes. Similar studies are projected for the muscle-specific (MM) and ubiquitous (BB) isozymes of creative phosphoskinase anticipating that the synthesis of the BB form will map elsewhere in the cycle than the protracted portion of G1. Experiments are also planned to reversibly protract G1 for different lengths of times with inhibitors to examine alternative switching mechanisms from the proliferative to the differentiative mode, and will be extended by a program to select its mutants blocked at different points in G1. The block to DNA synthesis and the stability of differentiation of muscle cell nuclei will also be examined after transfer to environments primed to support either DNA synthesis or transcription.